不同预处理方法对提取尿液脱落细胞mRNA质量的影响

目的 探索合适的处理尿标本的方法获得尿脱落细胞mRNA。方法 比较尿液容量、尿液标本冻存时间、处理方式对提取尿液脱落细胞RNA的产量与质量的影响,并进行qPCR的试验。结果 40 ml尿液标本组及10 ml尿液标本组RNA总量均值分别为886.94±222.93 ng及211.22±62.01 ng,两组A_260nm/A_280nm比值分别为1.80±0.10和1.77±0.09; 尿液标本量与RNA总量相关(t=3.604,P=0.002),不同尿液标本量RNA质量差异无统计学意义(t=0.708,P值>0.05)。实时离心提取及冰冻保存尿液7天后提取两种预处理方法RNA总量分别为886.94±945.84 ng及881.50±829.02 ng,A_260nm/A_280nm比值分别为1.80±0.10及1.80±0.87; RNA总量与A_260nm/A_280nm比值差异无统计学意义(t=-0.221～0.085,P值均>0.05)。直接冰冻尿液标本与TRIzol保存沉渣细胞两种预处理方法RNA总量分别为637.81±525.24 ng及639.86±535.42 ng,A_260nm/A_280nm比值分别为1.84±0.13及1.83±0.96; RNA总量与A_260nm/A_280nm比值差异无统计学意义(t=-0.47～0.293,P值均>0.05)。尿脱落细胞mRNA中可以检测到足细胞的标记蛋白WT-1,podocin和synaptopodin的基因表达。结论 尿液标本量是提取脱落细胞RNA量的关键因素,-80℃低温冻存尿标本7天或Trizol预处理冻存均对提取尿液沉渣细胞RNA数量和质量无影响。

Effects of Different Pretreatment Methods on mRNA Qualityand Quantity of Exfoliated Urinary Cells

Objective To explore a proper method to obtainexfoliated cells mRNA for detection in urine specimen.Methods Urine volume,different pretreating ways and frozen storage time on urinary sample were used to collect mRNA.A_260nm/A_280nm and A_260nm/A_280nm ratio,as well as qPCR were performed.Results Amount of mRNA was determinded by urine volume,but neitherA_260nm/A_280nm or A_260nm/A_280nm ratio wasrealated with urine volume.Compared with real-time extraction process,freezing urine samples for 7 days showed no statistical difference(P>0.05)in total RNA and A_260nm/A_280nm or A_260nm/A_280nm ratio.There was no significant difference in total RNA and A_260nm/A_280nm or A_260nm/A_280nm ratio between direct freezing urine samples and Trizol-treated urine exfoliated cells.Podocyte marker including WT-1,podocin and synaptopodin mRNAs were detected in urine sediment cells.Conclusion Quantity of mRNA was determinded by urine volume.Directly freezing urinesamples at -80 ℃ for 7 days or Trizol-treating urine sediment cells have no effect on lowing quality and quantity of urinary sediment mRNA.